The goal of this proposal is to gain insight into the relationship between glycolysis and cellular calcium homeostasis following myocardial ischemia and reperfusion. The applicant has found that inhibition of glycolytic flux during the first few minutes of reperfusion results in poor functional and metabolic recovery of ischemic/reperfused rabbit hearts, with marked and persistent elevation of free cytosolic Ca2+ concentration, despite administration of oxidative substrates. The applicant interprets these findings as suggesting that ATP may be functionally compartmentalized in the heart such that glycolytic ATP is functionally coupled to the SR Ca2+-ATPase and fuels the SR Ca2+ pump in cardiac and skeletal muscle. Studies with SR vesicles isolated from rabbit skeletal muscle will identify whether associated glycolytic enzymes are present, and in what specific activity, whether addition of glycolytic substrates and cofactors without exogenous ATP can drive the SR Ca2+-ATPase and support intravesicular transport of 45Ca, and whether evidence of functional "channeling" of glycolytic ATP and glycolytic intermediates exists in this system. The conditions under which glycolytic enzymes are solubilized from SR with loss of energetic support of 45Ca transport will be characterized. Isolated rabbit and rat cardiac myocytes will be studied to determine the comparative effects of inhibition of glycolysis (with iodoacetic acid or 2-deoxyglucose) vs oxidative phosphorylation (with cyanide) on SR Ca2+ transport, evaluated by changes in (Ca2+)i (indo-1 fluorescence) and cell shortening during cold contracture, rapid addition of caffeine, and elicitation of a staircase, and by the rate of decay of the Ca transient. Isolated perfused rabbit hearts will be studied with 13C-NMR to define the site and time course of the glycolytic inhibition that is observed early after reperfusion. SR isolated from ischemic/reperfused rabbit hearts will be examined to determine whether there is inhibition of one or more associated glycolytic enzymes.